br Introduction A woman is born with

Introduction A woman is born with approximately 295,000 oocytes arrested in the prophase of meiosis I, also commonly referred to as the germinal vesicle (GV) stage (Wallace and Kelsey, 2010). Unlike in mice whose oocytes start to grow and mature from the neonatal period, human oocytes stay quiescent in meiosis I until puberty or beyond (Smallwood and Kelsey, 2012). After puberty, 10–15 GV oocytes are recruited for growth and maturation during each menstrual cycle, of which only 1–2 reach the mature metaphase II (MII) stage soon before the ovulation, ready for fertilization, while the rest of the growing oocyte cohort degenerate. The genetic and epigenetic basis for the selection of dominant oocytes destined for ovulation and fertilization from the rest of the growing cohort is poorly understood (Surani, 2015). DNA methylation is a dynamic process during the growth and development of oocytes. It has been shown in mice that DNA methylation is erased in the primordial germ dabigatran etexilate during early embryonic development and re-established after birth as the oocytes start to grow and mature. The wave of DNA methylation erasure and re-establishment is repeated after fertilization in pre-implantation embryos (Smallwood and Kelsey, 2012). These waves of DNA methylation changes in oocytes and embryos are essential for resetting genomic potential, establishing the germline, and marking correct developmental genes (Bogdanovic et al., 2016; Hargan-Calvopina et al., 2016; Hon et al., 2013; von Meyenn and Reik, 2015). In humans, the process of DNA methylome erasure in primordial germ cells and in pre-implantation embryos was recently demonstrated in several genome-wide studies, and this process resembles what was previously described in mice, although some significant differences exist (Gkountela et al., 2015; Guo et al., 2014a, 2015; Smith et al., 2014; Tang et al., 2015; von Meyenn and Reik, 2015). The timing of de novo DNA methylation of oocytes after epigenetic erasure of parental marks in embryos is not only scientifically intriguing, but also presents a clinically important opportunity for guiding the efforts in developing in vitro maturation methods for fertility treatment. However, the DNA methylome establishment and maintenance during human oocyte growth and maturation beyond the early prenatal stage has not yet been illuminated, partly due to technical limitations of genome-wide studies in cells only available in very small numbers (Yu et al., 2015). Previous studies mapping the DNA methylome in human oocytes (Okae et al., 2014; Smith et al., 2014) had to pool a large number of oocytes in certain stages of development, and therefore were unable to specifically investigate DNA methylome variations among different oocyte maturation stages. Recent advances in single-cell bisulfite sequencing technologies (Farlik et al., 2015; Gravina et al., 2015, 2016; Schwartzman and Tanay, 2015; Smallwood et al., 2014) now enable DNA methylome analysis in small samples, down to the level of single cells.
Results
Discussion For the final analyses we merged all the samples from each maturation stage, which not only increased the genomic coverage significantly but also minimized the impact of patient heterogeneity on the final conclusion. However, individual characteristics, such as age or fertility status, and differences in ovarian stimulation from stimulated assisted reproductive technology cycles could still be factors that confound our findings. Nevertheless, our findings of stable CpG methylation during oocyte maturation is reassuringly consistent with an earlier study using unstimulated oocytes and in vitro maturation protocol, which found unchanged CpG methylation of four imprinted genes during in vitro oocyte maturation (Kuhtz et al., 2014). With the rare availability of human oocytes, especially MII oocytes, for research purposes a collective effort from multiple research teams is needed in the future to collect a sample large enough to address all the limitations of sample heterogeneity.